DE3448148C2 - - Google Patents

Description

The invention relates to the use of glycoproteins having a molecular weight of 5,000 to 300,000 and 18 to 38% by weight of protein produced by culturing of Coriolus versicolor (Fr.) Quel. to improve the Deformity of erythrocytes.

That of Coriolus versicolor (Fr.) Quel. (FERM-P No. 2412) derived glycoprotein is on Market already available as antitumor drug under the trademark Krestin.

The trunks Coriolus versicolor (Fr.) Quel. FERM P-No. 2412 and FERM P no. 2414 were from the Fermentation Research Institutes, Agency of Industrial Science and Technology at the American Type Culture Collection on 30. July 1979 under the ATCC no. 20547 and the ATCC no. 20545 deposited.

Because the glycoprotein has low mammalian toxicity and the intestinal microflora does not bother, one can containing the glycoprotein as the active ingredient pharmaceutical preparation over a long time Be administered over a period of time. In addition, the glycoprotein free from the risk of causing malformations and / or allergic reactions and therefore the glycoprotein an extremely safe (safe) substance represents.

The glycoprotein is one already known substance and described for example in Japanese Patent Publication No. 17149/1971,  36 322/1976, 14 274/1981, 14 276/1981, 39 288/1981 and the Japanese Patent Application 57-1 34 496, according to which Glycoprotein is obtained by culturing a Basidiomycete fungi species, which belongs to the genus Coriolus, Extract the thus greatly increased mycelium or fruiting bodies with hot water or an aqueous one Alkaline solution and removal of the low molecular weight substances with a molecular weight of less than 5000, being in this way in the form of an extract substance obtained contains about 18 to 38 wt .-% proteins and a molecular weight of 5,000 to 300,000, determined by the ultracentrifuge method.

Numerous pharmacological properties are in the Company publication "Outline of PSK", pages 28 to 30, described.

From DE-AS 26 59 808 also the use of this Glycoproteins known for fighting tumors.

That from the mycelia of Coriolus versicolor (Fr.) Quel. derived glycoprotein has a liver-brown color and a nitrogen content of 2 to 8%, in many cases of 3 until 6%. Various color reaction tests with the inventive Glycoprotein were performed the following results:

α- Naphthol-sulfuric acid reaction (Molish reaction) Purple Indole-sulfuric acid reaction (Dische reaction) brown Anthrone sulfuric acid reaction Greenish-blue Phenol-sulfuric acid reaction brown Tryptophan-sulfuric acid reaction Purple-Brown Lowry-Folin method blue Ninhydrin reaction after hydrochloric acid hydrolysis Greenish-blue

The molecular weight of the glycoprotein is 5000 to 300,000, measured using a  Ultracentrifugation method. The glycoprotein contains about 18 to 38 wt .-% proteins.

The saccharide portion of the glycoprotein consists mainly of β- D-glycan and the structure of the glycan residue is a branched structure having 1 → 3, 1 → 4 and 1 → 6 bonds. Of the amino acids that make up the protein portion of the glycoprotein, the amount of acidic amino acids, such as. As aspartic acid, glutamic acid and the like., And those of the neutral amino acids, such as valine, leucine and the like., Relatively large and the amount of basic amino acids, such as. As lysine, argenin and the like., Is relatively small. The glycoprotein is soluble in water and almost insoluble in hexane, benzene, chloroform, methanol and pyridine. The glycoprotein decomposes slowly at a temperature of about 120 ° C when heated.

As can be seen from the following Table I, mammalian toxicity is of the glycoprotein extremely low and it hardly causes any side effects in animals out. In particular, it is known as a very safe (safe) substance for living beings.  

Table I

The acute in the test for determining the above Toxicity value (LD⁵⁰ mg / kg) used those of strain ICR-JCL, 4 to 5 weeks after birth and with a body weight of 21 to 24 g. The in the Rats used in the same test were those from the strain Donryu, 4 to 5 weeks after birth and with a body weight from 100 to 150 g. The glycoprotein was in a dissolved in physiological saline and on each in administered according to Table I. After administration were the general symptoms, mortality and the body weight of each of the treated animals 7 days long watched and then they were killed and one Subjected to autopsy.  

As indicated in Table I, both in the case of Mice as well as in the case of the rats themselves at the maximum Dose that could be administered, no death detected, so that the glycoprotein for Living organisms extremely safe (safe) is up to such Just that the value for the LD₅₀ actually did not notice could be.

As a result of testing the physiological and pharmaceutical Properties of Coriolus versicolor (Fr.) Quel. derived glycoprotein was found to be the glycoprotein one of the deformability of erythrocytes increasing Activity in a mammal, and based on the present invention.

The invention relates to the use of glycoproteins having a molecular weight of 5,000 to 300,000 (determined by the ultracentrifuge method) and 18 to 38% by weight. protein content produced by cultivating Coriolus versicolor (Fr.) Quel., Extracting the on This strongly proliferated mycelia and fruiting bodies with hot water or aqueous alkali solution and removal the substances with a molecular weight of less than 5000 from the extract, to improve the deformability of erythrocytes.  

The glycoprotein has an activity Improvement of the deformation capacity of Erythrocytes are on and for treatment ischemic cerebral diseases.

The following are the pharmacological properties of the glycoprotein, and that the Deforming capacity of erythorocytes improving Activity.

In the erythorocytes of an animal, the Glycoprotein was administered, a decrease in the Hemolysis rate versus mechanical hemolytic Effect, d. H. in other words an improvement of the Deformity of the erythrocytes, found. The Blood flow in the cerebral ischemic condition was one Microcirculation and there an activity in relation to the Improvement of the deformability of the erythrocytes with the glycoprotein was detected was derived from the fact that the active ingredient, the glycoprotein, was able to control the blood circulation to facilitate. In addition, the glycoprotein was active with respect to the inhibition of death of the test animal as a result of thrombosis caused by the experimental animal administered arachidonic acid.

The glycoprotein is useful as a remedy  ischemic cerebral disease due to both its improving the deformability of erythrocytes Activity also due to his death as a result of a Thrombosis inhibitory activity. According to the present Invention is the glycoprotein preparation for improvement ischemic cerebral diseases, d. H. for the treatment of Cerebral thrombosis or ischemic diseases that accompany the cerebral infarction as a result of arteriosclerosis.

The administration of the preparation can be carried out as follows become:

By combined use for the improvement of ischemic Cerebral diseases with an agent for enlargement of the cerebral blood vessels, such. B. carbon dioxide, papaverine, Buphenin, Isoxsuprin, Hexobendin, Cyclandelate, ATP, ADP, adenosine phosphate, acetazolamide, pyritinol, raubasin, an aqueous hypertonic glucose solution and the like, a combination effect is to be expected. In addition, can it also in Used in combination with an anti-arteriosclerotic agent become.

The glycoprotein may be oral or parenteral, preferably orally, administered to humans. The oral administration includes sublingual administration and parenteral administration includes the subcutaneous  Injection, intramuscular injection, intravenous Injection and instillation. The effective amount of administration of the glycoprotein depends on the species, the Age, individual differences and disease state of the patient, in the case of treatment of human patients however, the daily dose is 10 to 1000 mg, preferably 200 to 600 mg per kg of body weight, the is divided equally into 1 to 3 servings to 1 to be administered 3 times a day.

In the case of oral administration, the glycoprotein may be present in the usual forms in galenics, for example in the form of a tablet, granules, a powder and a capsule, it can be in liquid Form, for example in the form of solutions, suspensions, Emulsions and syrups, they can be in the form of Mixtures are present, which are used after shaking be, or it may be in a fixed form, the after dissolving in sterilized water containing no containing pyretic substance is used. Is it lying in solid form, it may contain conventional additives, such as z. As binders, diluents, lubricants, disintegrators, Wetting agents; in liquid form, it can be commonly used Contains additives and preservatives. In case of a Injection, the glycoprotein preparation may contain further additives, such as As stabilizers, buffers, preservatives and Isotonic agents are included, and the product is after the filling into a dosage unit ampoule or in a conventional container placed on the market.

The invention will be more apparent from the following examples explained without, however, limiting it to.

Example 1 Production of the glycoprotein

In 4 l of an aqueous 0.1 N sodium hydroxide solution were 200 g dried mycelium of Coriolus versicolor (Fr.) Quel. ATCC 20545 with a moisture content of 8.8% and an approximate nitrogen content of 2.5% and the mycelia were added with stirring Temperature extracted from 90 to 95 ° C for 1 h and then The mixture was cooled to below 50 ° C and after the Adjusting the pH of the cooled in this way Mixture to 7.0 with an aqueous 1 N hydrochloric acid solution the dissolved material was filtered by suction removed from the mixture and removed in this way solid material was washed with 500 ml of water. The mixture of the filtrate and the washing waters, the 4.2 l was using a desktop ultrafilter the company Amicon Inc. (equipped with the ultrafiltration membrane PM-5) with stirring and cooling under a working pressure of 1.5 kg / mc² at 10 ° C ultrafiltration subjected, whereby the low-molecular substances with a molecular weight of less than 5000 were removed, after which it was concentrated, so that 300 ml of the treated aqueous extract. The watery Extract was subjected to freeze-drying, wherein about 26.6 g of a powdery substance with a liver brown color in a yield of 13%, based on the mycelium, received. The obtained in this way powdery substance had a moisture content of 7.5% and an elemental analysis composition of 40.5% carbon, 6.2% hydrogen, 5.8% nitrogen and rest oxygen. The powdery substance was in Water slightly soluble.  

The powdery substance had an activity with respect to on the inhibition of propagation of the transplanted Sarcomas 180 of up to 90% when administered intraperitoneally transplanted mouse, and up to 65%, when administered orally to the transplanted mouse, on.

Subsequent Example 1a) Production of the glycoprotein

In 4 l of an aqueous 0.1 N sodium hydroxide solution were 200 g dried mycelium of Coriolus versicolor (Fr.) Quel. ATCC 20547 with a moisture content of 8.8% and an approximate nitrogen content of 2.5% and the mycelia were added with stirring Temperature extracted from 90 to 95 ° C for 1 h and then The mixture was cooled to below 50 ° C and after the Adjusting the pH of the cooled in this way Mixture to 7.0 with an aqueous 1 N hydrochloric acid solution the dissolved material was filtered by suction removed from the mixture and removed in this way solid material was washed with 500 ml of water. The mixture of the filtrate and the washing waters, the 4.1 l was obtained using a desktop ultrafilter the company Amicon Inc. (equipped with the ultrafiltration membrane PM-5) with stirring and cooling under a working pressure of 1.5 kg / mc² at 10 ° C ultrafiltration subjected, whereby the low-molecular substances with a molecular weight of less than 5000 were removed, after which it was concentrated to give 280 ml of the treated aqueous extract. The watery Extract was subjected to freeze-drying, wherein About 28.3 g of a powdery substance with a liver-brown color in a yield of 14%, based on the mycelium, received. The obtained in this way powdery substance had a moisture content of 7.3% and an elemental analysis composition of 41.2% carbon, 6.1% hydrogen, 5.8% nitrogen and rest oxygen. The powdery substance was in Water slightly soluble.

The powdery substance had an activity with respect to on the inhibition of propagation of the transplanted Sarcoma 180 of up to 95% when intraperitoneally transplanted mouse, and up to 68%, when administered orally to the transplanted mouse, on.

example 2 Activity relating to the improvement of the deformability of erythrocytes

The glycoprotein was orally or intraperitoneally administered to each of six groups of Wistar rats, each group consisting of 5 animals, at a dose of 10-1000 mg / kg. Three hours after the administration, the blood was taken out of each rat and added to 10 times the weight of a physiological saline solution. After stirring the mixture in a blender, whereby the erythrocytes in the mixture were mechanically hemolyzed, the mixture was subjected to centrifugal separation. The amount of hemoglobin (A _i ) in the resulting supernatant liquid was measured colorimetrically. The same procedures were carried out except that dilute water was used instead of the physiological saline solution, the amount of hemoglobin (B _i ) in the supernatant liquid was obtained, and the extent of hemolysis (H _i ) was calculated using the following formula:

The extent of hemolysis obtained in the blood of the control rat administered with distilled water in place of glycoprotein was calculated by the same method as H _c above.

The relative rate of hemolysis was calculated using the following formula using the mean of H _i and H _c :

The results obtained are shown in the following Table II after classification in the route of administration and the dosage rate indicated.

As can be seen from Table II, there was a decrease in relative hemolysis rate detected in the groups that Glycoprotein regardless of the route of administration of the glycoprotein. The administration of the Glycoproteins thus leads to an improvement Deformation capacity of erythrocytes.  

Table II

Activity with respect to the reduction of the relative degree of hemolysis

example 3 Formulation of a pharmaceutical preparation

Capsules, each containing 330 mg of the glycoprotein were made by filling of hard capsules # 0 with the glycoprotein as it existed using an automatic filling device negative pressure.

Claims (1)

Use of glycoproteins with a molecular weight from 5000 to 300,000 (determined by the ultracentrifuge method) and 18 to 38% by weight protein content, made by cultivating Coriolus versicolor (FR.) Source: Extract the greatly increased mycelium in this way or fruiting bodies with hot water or an aqueous one Alkaline solution and removing the substances with a molecular weight of less than 5,000 from the Extract, to improve the deformability of erythrocytes.